The long-term objective of this project is to characterize the molecular pathways responsible for bladder smooth muscle development, function, and pathogenesis. Disorders of the urinary bladder affect millions of people in the United States annually and have a devastating impact on quality of life and morbidity (1, 2). In spite of the large number of people affected and the substantial financial impact these disorders have on our community, the molecular pathway(s) controlling bladder development, function, and pathogenesis are not well understood. Studies in our lab have identified a unique murine model for studying bladder dysmorphogenesis (3). These animals, designated mgb for megabladder, develop a lower urinary tract obstruction in utero due to a lack of detrusor smooth muscle development (3). Complementation analysis, expression studies, and qPCR have indicated that myocardin (Myocd) is responsible for the bladder smooth muscle defect in the mgb mouse. Based on observations from these studies we hypothesize that bladder smooth muscle development is highly dependent and susceptible to altered levels of Myocd expression. To address this hypothesis, we propose to: 1) Examine the transcriptional complex formed by Myocardin on a prototypic smooth muscle specific promoter in both wild type and mgb-/- bladders. We will use EMSA analysis to compare the interactions of normal (WT) and mutant (mgb) bladder nuclear proteins with a prototypic smooth muscle-specific promoter element at three key developmental time points in smooth muscle differentiation in order to characterize how the loss of Myocd in our mgb animals impacts transcriptional protein complex formation on bladder smooth muscle specific genes. Mass Spectrometry will be used to determine the precise protein composition of the complex(es) identified by EMSA necessary for bladder smooth muscle gene expression. 2) Recapitulate and rescue the mgb phenotype in vitro by altering Myocardin expression. We will use in vitro whole organ explant cultures to characterize and manipulate bladder smooth muscle in order to assess if altering Myocd expression is sufficient to influence bladder smooth muscle development. PUBLIC HEALTH RELEVANCE: Disorders of the urinary bladder and its associated structures affect both sexes of all ages and ethnic backgrounds. The mgb mouse model provides us with an unparalleled resource to specifically study bladder smooth muscle development and bladder dysmorphogenesis. The studies proposed here with the mgb mouse model will provide a basis for the development of new methods of detection, prognosis, and therapeutic strategies, including tissue engineering, to treat a variety of bladder diseases and dysfunction.